Attenuated pestiviruses

ABSTRACT

This invention relates to attenuated pestiviruses characterised in that their enzymatic activity residing in glycoprotein E RNS  is inactivated, methods of preparing, using and detecting these.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for attenuatingpestiviruses by inactivating the ribonuclease activity (RNase activity)residing in glycoprotein E^(RNS). The invention also relates topestiviruses attenuated according to the invention, nucleic acids forpreparing such pestiviruses, vaccines and pharmaceutical compositionscomprising the attenuated pestiviruses of the invention. The inventionfurther relates to methods for distinguishing between the attenuatedviruses of the invention and pathogenic viruses.

BACKGROUND OF THE INVENTION

[0002] Pestiviruses are causative agents of economically importantdiseases of animals in many countries worldwide. Presently known virusisolates have been grouped into three different species which togetherform one genus within the family Flaviviridae.

[0003] I Bovine viral diarrhea virus (BVDV) causes bovine viral diarrhea(BVD) and mucosal disease (MD) in cattle (Baker, 1987; Moennig andPlagemann, 1992; Thiel et al., 1996).

[0004] II Classical swine fever virus (CSFV), formerly named hog choleravirus, is responsible for classical swine fever (CSF) or hog cholera(HC) (Moennig and Plagemann, 1992; Thiel et al., 1996).

[0005] III Border disease virus (BDV) is typically found in sheep andcauses border disease (BD). Symptoms similar to MD in cattle have alsobeen described to occur after intrauterine infection of lambs with BDV(Moennig and Plagemann, 1992; Thiel et al., 1996).

[0006] An alternative classification of pestiviruses is provided byBecher et al. (1995) or others.

[0007] Pestiviruses are small enveloped viruses with a single strandedRNA genome of positive polarity lacking both 5′ cap and 3′ poly(A)sequences. The viral genome codes for a polyprotein of about 4000 aminoacids giving rise to final cleavage products by co- andposttranslational processing involving cellular and viral proteases. Theviral proteins are arranged in the polyprotein in the orderNH₂-N^(pro)-C-E^(RNS)-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (Rice,1996). Protein C and the glycoproteins E^(RNS), E1 and E2 representstructural components of the pestivirus virion (Thiel et al., 1991). E2and to a lesser extent E^(RNS) were found to be targets for antibodyneutralization (Donis et al., 1988; Paton et al., 1992; van Rijn et al.,1993; Weiland et al., 1990,1992). E^(RNS) lacks a membrane anchor and issecreted in considerable amounts from the infected cells; this proteinhas been reported to exhibit RNase activity (Hulst et al., 1994;Schneider et al., 1993; Windisch et al., 1996). The function of thisenzymatic activity for the viral life cycle is presently unknown. In thecase of a CSFV vaccine strain experimental destruction of the RNase bysite directed mutagenesis has been reported to result in acytopathogenic virus that has growth characteristics in cell cultureequivalent to wild type virus (Hulst et al., 1998). The enzymaticactivity depends on the presence of two stretches of amino acidsconserved between the pestivirus E^(RNS) and different known RNases ofplant and fungal origin. Both of these conserved sequences contain ahistidine residue (Schneider et al., 1993). Exchange of each of theseresidues against lysine in the E^(RNS) protein of a CSFV vaccine strainresulted in the destruction of RNase activity (Hulst et al., 1998).Introduction of these mutations into the genome of the CSFV vaccinestrain did not influence viral viability or growth properties but led toa virus exhibiting a slightly cytopathogenic phenotype (Hulst et al.,1998).

[0008] Vaccines comprising attenuated or killed viruses or viralproteins expressed in heterologous expression systems have beengenerated for CSFV and BVDV and are presently used. The structural basisof the attenuation of these viruses used as life vaccines is not known.This leads to the risk of unpredictable revertants by backmutation orrecombination subsequent to vaccination. On the other hand, the efficacyof inactivated vaccines or heterologously expressed viral proteins(subunit vaccines) in the induction of immunity is rather low.

[0009] In general, live vaccines with defined mutations as a basis forattenuation would allow to avoid the disadvantages of the presentgeneration of vaccines. Potential targets for attenuating mutations inpestiviruses are not available at present.

[0010] A further advantage of said attenuating mutations lies in theirmolecular uniqueness which allows to use them as distinctive labels foran attenuated pestiviruses and to distinguish them from pestivirusesfrom the field.

[0011] Because of the importance of an effective and safe as well asdetectable prophylaxis and treatment of pestiviral infections, there isa strong need for live and specifically attenuated vaccines with a highpotential for induction of immunity as well as a defined basis ofattenuation which can also be distinguished from pathogenicpestiviruses.

[0012] Therefore, the technical problem underlying the present inventionis to provide specifically attenuated and detectably labeledpestiviruses for use as live attenuated vaccines with a high efficiencyfor the induction of immunity which, as a result of this method, canalso be distinguished from pathogenic pestiviruses from the field.

DISCLOSURE OF THE INVENTION

[0013] The solution to the above technical problem is achieved by thedescription and the embodiments characterized in the claims.

[0014] It has surprisingly been found that pestiviruses can bespecifically attenuated by the inactivation of the RNase activityresiding in glycoprotein E^(RNS).

[0015] The attenuated pestiviruses now provide live vaccines of highimmunogenicity.

[0016] Therefore, in one aspect the present invention provides a livevaccine comprising a pestivirus, wherein the RNase activity residing inglycoprotein E^(RNS) is inactivated.

[0017] The term “vaccine” as used herein refers to a pharmaceuticalcomposition comprising at least one immunologically active componentthat induces an immunological response in an animal and possibly but notnecessarily one or more additional components that enhance theimmunological activity of said active component. A vaccine mayadditionally comprise further components typical to pharmaceuticalcompostions. The immunologically active component of a vaccine maycomprise complete live organisms in either its original form or asattenuated organisms in a so called modified live vaccine (MLV) ororganisms inactivated by appropriate methods in a so called killedvaccine (KV). In another form the immunologically active component of avaccine may comprise appropriate elements of said organisms (subunitvaccines) whereby these elements are generated either by destroying thewhole organism or the growth cultures of such organisms and subsequentpurification steps yielding in the desired structure(s), or by syntheticprocesses induced by an appropriate manipulation of a suitable systemlike, but not restricted to bacteria, insects, mammalian or otherspecies plus subsequent isolation and purification procedures or byinduction of said synthetic processes in the animal needing a vaccine bydirect incorporation of genetic material using suitable pharmaceuticalcompositions (polynucleotide vaccination). A vaccine may comprise one orsimultaneously more than one of the elements described above.

[0018] Additional components to enhance the immune response areconstituents commonly referred to as adjuvants, like e.g.aluminiumhydroxide, mineral or other oils or ancillary molecules addedto the vaccine or generated by the body after the respective inductionby such additional components, like but not restricted to interferons,interleukins or growth factors.

[0019] A “pharmaceutical composition” essentially consists of one ormore ingredients capable of modifying physiological e.g. immunologicalfunctions of the organism it is administered to, or of organisms livingin or on its surface like but not restricted to antibiotics orantiparasitics, as well as other constituents added to it in order toachieve certain other objectives like, but not limited to, processingtraits, sterility, stability, feasibility to administer the compositionvia enteral or parenteral routes such as oral, intranasal, intravenous,intramuscular, subcutaneous, intradermal or other suitable route,tolerance after administration, controlled release properties.

[0020] A vaccine of the invention refers to a vaccine as defined above,wherein one immunologically active component is a pestivirus or ofpestiviral origin.

[0021] The term “live vaccine” refers to a vaccine comprising a living,in particular, a living viral active component.

[0022] The term “pestivirus” as used herein refers to all pestiviruses,characterized by belonging to the same genus as BVDV, CSFV and BDVwithin the family Flaviviridae and by their expression of glycoproteinE^(RNS). Of course, said term also refers to all pestiviruses ascharacterized by Becher et al. (1995) or others that expressglycoprotein E^(RNS). “RNase activity” as used herein refers to theability of the glycoprotein E^(RNS) to hydrolyze RNA.

[0023] It should be noted that the term glycoprotein E0 is often usedsynonymously to glycoprotein E^(RNS) in publications.

[0024] The term “inactivation of the RNase activity residing in saidglycoprotein” refers to the inability or reduced capability of amodified glycoprotein E^(RNS) to hydrolyze RNA as compared to theunmodified wild type of said glycoprotein E^(RNS).

[0025] Inactivation of the RNase activity residing in glycoproteinE^(RNS) can be achieved by deletions and/or mutations of at least oneamino acid of said glycoprotein as demonstrated herein and by Hulst etal. (1998). Therefore, in a preferred embodiment the present inventionrelates to live vaccines, wherein said RNase activity is inactivated bydeletions and/or mutations of at least one amino acid of saidglycoprotein.

[0026] It has been shown that the glycoprotein E^(RNS) forms adisulfide-bonded homodimer of about 97 kD, wherein each monomer consistsof 227 amino acids corresponding to the amino acids 268 to 494 of theCSFV polyprotein as described by Rümenapf et al. (1993). The first 495amino acids as expressed by the Alfort strain of CSFV are shown in FIG.1 for reference purpose only. The genome sequence of the Alfort strainof CSFV is available in the GenBank/EMBL data library under accessionnumber J04358; alternatively, the amino acid sequence for the BVDVstrain CP7 can be accessed in the GenBank/EMBL data library (accessionnumber U63479). Two regions of amino acids are highly conserved inglycoprotein E^(RNS) s as well as in some plant and fungal RNase-activeproteins (Schneider et al., 1993). These two regions are of particularimportance to the RNase enzymatic activity. The first region consists ofthe region at the amino acids at position 295 to 307 and the secondregion consists of the amino acids at position 338 to 357 of said viralpolyprotein as exemplified by FIG. 1 for the Alfort strain of CSFV(numbering according to the published deduced amino acid sequence ofCSFV strain Alfort (Meyers et al., 1989). The amino acids of particularimportance to the RNase activity as mentioned above are by no meanslimited to the exact position as defined for the Alfort strain of CSFVbut are simply used in an exemplary manner to point out the preferredamino acids being at that position or corresponding to that position inother strains such as found in BVDV, BDV and pestiviruses in generalsince they are highly conserved. For pestiviruses other than the CSFVAlfort strain the numbering of the positions of the preferred aminoacids is often different but an expert in the field of the molecularbiology of pestiviruses will easily identify these preferred amino acidsby their position relative to the highly conserved amino acids of saidglycoprotein. In one particular non-limiting example, the position ofCSFV Alfort 346 is identical to position 349 of BVDV strain cp7.

[0027] As a consequence, the present invention relates in a morepreferred embodiment to a vaccine of the invention, wherein saidinactivating deletions and/or mutations are located at the amino acidsat position 295 to 307 and/or position 338 to 357, as described in FIG.1 for the CSFV Alfort strain in an exemplary manner or correspondingthereto in other strains, of said glycoprotein.

[0028] In a very preferred embodiment the present invention disclosesthat the inactivation of said RNase activity by deletion or mutation ofthe amino acid at position 346 of said glycoprotein leads toparticularly useful live vaccines. Therefore, the present inventionrelates to vaccines according to the invention, wherein said Rnaseactivity is inactivated by deletion or mutation of the amino acid atposition 346, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other strains, of saidglycoprotein.

[0029] The present invention demonstrates that pestiviruses are viableand code for an E^(RNS) protein without RNase activity when thehistidine residue at position 346 of the viral polyprotein (numberingaccording to the published sequence of CSFV Alfort/Tübingen (Meyers etal., 1989)), which represents one of the conserved putative active siteresidues of the E^(RNS) RNase, is deleted. It has also been demonstratedfor this invention that the deletion of the respective histidine in theE^(RNS) of a BVD pestivirus (position 349, numbered according to thesequence of BVDV CP7 GenBank/EMBL data library (accession numberU63479)) results in a viable virus in which the E^(RNS) glycoprotein haslost the RNase activity. In contrast to point mutations changing oneamino acid into another, a deletion mutant is generally much more stablewith respect to revertants. Infection of pigs with a mutant of thepathogenic CSFV Alfort/Tübingen expressing E^(RNS) with this deletiondid not lead to fever or other typical clinical signs of CSFV infectionswhereas the infection with wild type virus resulted in fever, diarrhea,anorexia, apathy, depletion of B-cells and central nervous disorders.These pigs were killed in a moribund stage showing severe hemorrhages inthe skin and internal organs 14 days post inoculation. The pigs infectedwith the mutant did neither show viremia nor B-cell depletion as testedon days 3, 5, 7, 10, 14 post infection while CSFV was easily isolatedfrom blood samples derived from the pigs inoculated with wild typevirus. The deletion mutant apparently replicated in the animals asindicated by the induction of neutralizing antibodies (see Example 3,Table 3c). The immune response to the mutant virus was sufficient topermit to survive a lethal challenge with 2×10⁵ TCID₅₀ of the highlypathogenic infection with the CSFV strain Eystrup (König, 1994) which isheterologous to the Alfort strain. Moreover, the tested animalsdisplayed no typical clinical signs for CSFV infection like fever,diarrhea, hemorrhages, B-cell depletion or anorexia after the challengeinfection. This data demonstrates that infection of pigs with thedeletion mutant induces an immune response sufficient for protectionagainst a stringent challenge.

[0030] Therefore, in a most preferred embodiment, the invention relatesto vaccines according to the invention, wherein said RNase activity isinactivated by the deletion of the histidine residue at position 346, asdescribed in FIG. 1 for the CSFV Alfort strain in an exemplary manner orcorresponding thereto in other strains, of said glycoprotein.

[0031] In a further most preferred embodiment, the invention relates toBVDV vaccines according to the invention, wherein said RNase activity isinactivated by the deletion of the histidine residue at position 346, asdescribed in FIG. 1 for the CSFV Alfort strain in an exemplary manner orcorresponding thereto in other BVDV strains, of said glycoprotein.

[0032] In another aspect the present invention relates to attenuatedpestiviruses, wherein the RNase activity residing in glycoproteinE^(RNS) is inactivated by deletions and/or mutations of at least oneamino acid of said glycoprotein with the proviso that the amino acids atposition 297 and/or 346 of said glycoprotein as described in FIG. 1 forCSFV are not lysine. A recombinant pestivirus, wherein amino acids atposition 297 and/or 346 of said glycoprotein are lysine has beendescribed by Hulst et al. in 1998. These particular pestivirusesdemonstrated cytopathic effects in swine kidney cells. Up to now, therehas been total unawareness of the surprising and innovative attenuatingfeature due to the inactivation of the E^(RNS) enzymatic activity.

[0033] In a preferred embodiment for the reasons stated above forvaccines the present invention also relates to pestiviruses according tothe invention, wherein said RNase activity is inactivated by deletionsand/or mutations located at the amino acids at position 295 to 307and/or position 338 to 357, as described in FIG. 1 for the CSFV Alfortstrain in an exemplary manner or corresponding thereto in other strains,of said glycoprotein.

[0034] In a more preferred embodiment for the reasons stated above forvaccines the present invention also relates to pestiviruses of theinvention, wherein said RNase activity is inactivated by deletion ormutation of the amino acid at position 346, as described in FIG. 1 forthe CSFV Alfort strain in an exemplary manner or corresponding theretoin other strains, of said glycoprotein.

[0035] In a most preferred embodiment for the reasons stated above forvaccines the present invention also relates to pestiviruses, whereinsaid RNase activity is inactivated by the deletion of the histidineresidue at position 346, as described in FIG. 1 for the CSFV Alfortstrain in an exemplary manner or corresponding thereto in other strains,of said glycoprotein.

[0036] In a further most preferred embodiment, the present inventionrelates to BVDV pestiviruses, wherein said RNase activity is inactivatedby the deletion of the histidine residue at position 346, as describedin FIG. 1 for the CSFV Alfort strain in an exemplary manner orcorresponding thereto in other BVDV strains, of said glycoprotein.

[0037] The attenuated pestiviruses and active components of the vaccinesof the present invention can easily be prepared by nucleicacid-modifying recombinant techniques resulting in the expression of amutant amino acid sequence in glycoprotein E^(RNS). Therefore, a furtheraspect of the present invention relates to nucleic acids coding for aglycoprotein E^(RNS), wherein the RNase activity residing in saidglycoprotein is inactivated by deletions and/or mutations of at leastone amino acid of said glycoprotein with the proviso that the aminoacids at position 297 and/or 346 of the glycoprotein as described inFIG. 1 for the CSFV Alfort strain are not lysine.

[0038] In a preferred embodiment the present invention relates, forreasons as mentioned above, to nucleic acids according to the invention,wherein said RNase activity is inactivated by deletions and/or mutationsthat are located at the amino acids at position 295 to 307 and/orposition 338 to 357, as described in FIG. 1 for the CSFV Alfort strainin an exemplary manner or corresponding thereto in other strains, ofsaid glycoprotein.

[0039] In a more preferred embodiment the present invention relates, forreasons as mentioned for vaccines, to nucleic acids according to theinvention, wherein said RNase activity is inactivated by deletion ormutation of the amino acid at position 346, as described in FIG. 1 forthe CSFV Alfort strain in an exemplary manner or corresponding theretoin other strains, of said glycoprotein.

[0040] In a most preferred embodiment the present invention relates tonucleic acids according to the invention, wherein said RNase activity isinactivated by the deletion of the histidine residue at position 346, asdescribed in FIG. 1 for the CSFV Alfort strain in an exemplary manner orcorresponding thereto in other strains, of said glycoprotein.

[0041] In a further most preferred embodiment the present inventionrelates to BVDV nucleic acids according to the invention, wherein saidRNase activity is inactivated by the deletion of the histidine residueat position 346, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other BVDV strains, of saidglycoprotein.

[0042] Nucleotides, e.g. DNA or RNA, are also useful for preparing DNA-,RNA- and/or vector-vaccines. In these vaccines, the nucleotides areapplied directly to the animal or indirectly via vectors other than theoriginal virus. Nucleotide vaccines and vector vaccines are well knownfrom the present state of the art and will not be elaborated further.

[0043] In a further aspect, the present invention relates to the use ofnucleic acids of the present invention for preparing nucleotide- and/orvector-vaccines.

[0044] The vaccines, attenuated pestiviruses, and/or nucleic acidsaccording to the invention are particularly useful for the preparationof a pharmaceutical composition.

[0045] In consequence, a further aspect of the present invention relatesto pharmaceutical compositions comprising a vaccine according to theinvention, and/or a pestivirus according to the invention, and/or anucleotide sequence according to the invention. One non-limiting exampleof such a pharmaceutical composition, solely given for demonstrationpurposes, could be prepared as follows: Cell culture supernatant of aninfected cell culture is mixed with a stabilizer (e.g. spermidine and/orBSA (bovine serum albumin)) and the mixture is subsequently lyophilizedor dehydrated by other methods. Prior to vaccination, said mixture isthen rehydrated in aquous (e.g. saline, PBS (phosphate buffered saline))or non-aquous solutions (e.g. oil emulsion, aluminum-based adjuvant).

[0046] An additional aspect of the present invention relates to a methodof attenuation for pestiviruses. The invention provides a unique andunexpected method for attenuating pestiviruses characterized in that theRNase activity residing in glycoprotein E^(RNS) is inactivated.

[0047] The specifically attenuated pestiviruses are especially usefulfor the preparation of vaccines. Therefore, in a further additionalaspect the present invention relates to methods for producing aspecifically attenuated pestivirus vaccine characterized in that theRnase activity residing in glycoprotein E^(RNS) is inactivated.

[0048] The inactivation of the RNase activity residing in glycoproteinE^(RNS) provides a surprising and new method for detectably labelingpestiviruses. In a further aspect the present invention provides amethod for detectably labeling pestiviruses characterized in that theRNase activity residing in glycoprotein E^(RNS) is inactivated. Thefeature of absence of RNase activity residing in the glycoproteinE^(RNS) of pestiviruses of the invention now enables for detectablylabeling these pestiviruses. Labeled and unlabeled pestiviruses or theE^(RNS) secreted from pestivirus infected cells in body fluids canclearly be distinguished by the absence or presence of RNase activity ofthe glycoproteins E^(RNS) upon isolation and assaying such enzymaticactivity.

[0049] For pestiviruses inactivated in their RNase activity residing inglycoprotein E^(RNS) by deletion and/or mutation, a number of othertechniques can be used. Such pestiviruses can easily be detected becauseof the structural consequences resulting from such deletions and/ormutations. For example, the sequence difference of the nucleic acidsequence of altered glycoprotein E^(RNS) is detectable by nucleic acidsequencing techniques or PCR-techniques (polymerase-chain reaction) asdemonstrated in example 8; the altered protein sequence can be detectedby specific monoclonal antibodies, that do not recognize unalteredproteins. Vice versa, it is also possible to detect the altered andthereby structurally labeled proteins by the absence of binding tospecific monoclonal antibodies that recognize unaltered glycoproteinsE^(RNS) under the proviso that the presence of pestiviruses can beestablished otherwise. And, of course, the deletions and/or mutationsabrogating the RNase activity in the labeled viruses will result indifferent immune responses in animals when compared to the responsesresulting from unlabeled pestivirus infections.

[0050] A preferred embodiment for all aspects referring to methods forattenuating pestiviruses, methods for producing a specificallyattenuated pestivirus vaccine and methods for detectably labelingpestiviruses according to the invention are those methods relating tothe inactivation of the glycoprotein E^(RNS), wherein said RNaseactivity is inactivated by deletions and/or mutations of at least oneamino acid of said glycoprotein.

[0051] A more preferred embodiment for all aspects referring to methodsfor attenuating pestiviruses, methods for producing a specificallyattenuated pestivirus vaccine and methods for detectably labelingpestiviruses according to the invention are those methods relating tothe inactivation of the glycoprotein E^(RNS), wherein said deletionsand/or mutations are located at the amino acids at position 295 to 307and/or position 338 to 357, as described in FIG. 1 for the CSFV Alfortstrain in an exemplary manner or corresponding thereto in other strains,of said glycoprotein.

[0052] A very preferred embodiment for all aspects referring to methodsfor attenuating pestiviruses, methods for producing a specificallyattenuated pestivirus vaccine and methods for detectably labelingpestiviruses according to the invention are those methods relating tothe inactivation of the glycoprotein E^(RNS), wherein said RNaseactivity is inactivated by deletion or mutation of the amino acid atposition 346, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other strains, of saidglycoprotein.

[0053] A most preferred embodiment for all aspects referring to methodsfor attenuating pestiviruses, methods for producing a specificallyattenuated pestivirus vaccine and methods for detectably labelingpestiviruses according to the invention are those methods relating tothe inactivation of the glycoprotein E^(RNS), wherein said RNaseactivity is inactivated by the deletion of the histidine residue atposition 346, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other strains, of saidglycoprotein.

[0054] The present invention provides vaccines and or otherpharmaceutical compositions which are particularly useful for theprophylaxis and treatment of pestivirus infections in animals.Therefore, a further aspect of the present invention relates to methodsfor the prophylaxis and treatment of pestivirus infections in animalscharacterized in that a vaccine according to the invention or anotherpharmaceutical composition according to the invention is applied to ananimal in need of such prophylaxis or treatment.

[0055] In a further aspect the present invention provides a process forthe preparation of specifically attenuated pestiviruses characterized inthat the RNase activity residing in glycoprotein E^(RNS) is inactivated.

[0056] In one aspect the present invention provides a process for thepreparation of specifically labeled pestiviruses characterized in thatthe RNase activity residing in glycoprotein E^(RNS) is inactivated.

[0057] A preferred embodiment for all aspects referring to a process forthe preparation of specifically attenuated pestiviruses, a process forthe preparation of specifically labeled pestiviruses according to theinvention are those processes relating to the inactivation of theglycoprotein E^(RNS), wherein said RNase activity is inactivated bydeletions and/or mutations of at least one amino acid of saidglycoprotein.

[0058] A more preferred embodiment for all aspects referring to aprocess for the preparation of specifically attenuated pestiviruses, aprocess for the preparation of specifically labeled pestivirusesaccording to the invention are those processes relating to theinactivation of the glycoprotein E^(RNS), wherein said deletions and/ormutations are located at the amino acids at position 295 to 307 and/orposition 338 to 357, as described in FIG. 1 for the CSFV Alfort strainin an exemplary manner or corresponding thereto in other strains, ofsaid glycoprotein.

[0059] A very preferred embodiment for all aspects referring to aprocess for the preparation of specifically attenuated pestiviruses, aprocess for the preparation of specifically labeled pestivirusesaccording to the invention are those processes relating to theinactivation of the glycoprotein E^(RNS), wherein said Rnase activity isinactivated by deletion or mutation of the amino acid at position 346,as described in FIG. 1 for the CSFV Alfort strain in an exemplary manneror corresponding thereto in other strains, of said glycoprotein.

[0060] A most preferred embodiment for all aspects referring to aprocess for the preparation of specifically attenuated pestiviruses, aprocess for the preparation of specifically labeled pestivirusesaccording to the invention are those processes relating to theinactivation of the glycoprotein E^(RNS), wherein said RNase activity isinactivated by the deletion of the histidine residue at position 346, asdescribed in FIG. 1 for the CSFV Alfort strain in an exemplary manner orcorresponding thereto in other strains, of said glycoprotein.

[0061] The vaccines or other pharmaceutical compositions of the presentinvention are useful for the prophylaxis and treatment of pestivirusinfections in animals.

[0062] Therefore, in one aspect the present invention relates to the useof a vaccine according to the invention for the prophylaxis andtreatment of pestivirus infections in animals. In a further aspect thepresent invention relates to the use of a pharmaceutical compositionaccording to the invention for the prophylaxis and treatment ofpestivirus infections in animals.

[0063] Pestiviruses and/or nucleic acids according to the invention areuseful active components of a pharmaceutical composition or a vaccine.Therefore, the present invention relates in a further aspect to the useof a pestivirus of the invention and/or a nucleic acid of the inventionfor the preparation of a vaccine or a pharmaceutical composition.

[0064] As mentioned above the inactivation of the RNase activityresiding in glycoprotein E^(RNS) provides a surprising and new methodfor labeling pestiviruses.

[0065] As a consequence one aspect of the present invention relates tomethods for distinguishing the detectably labeled pestiviruses accordingto the invention from unlabeled and possibly pathogenic pestiviruses.Such methods are especially useful for tracing the efficacy of labeledpestiviruses in animals. A vaccine treated animal will provelabel-positive after obtaining a sample of such animal and assaying forsaid label. Unlabeled animals and especially unlabeled animals thatprove pestivirus positive can be immediately separated, isolated orslaughtered to remove the imminent danger of spreading the pathogenicinfection to other animals.

[0066] The present invention provides a method for detectably labelingpestiviruses characterized in that the RNase activity residing inglycoprotein E^(RNS) is inactivated. This feature of absence of RNaseactivity residing in the glycoprotein E^(RNS) of pestiviruses of theinvention now enables for detectably labeling these pestiviruses. As aresult labeled and unlabeled pestiviruses can clearly be distinguishedby the absence or presence of RNase activity of the glycoprotein E^(RNS)upon isolation and assaying such enzymatic activity. The determinationof presence or absence of this enzymatic activity upon obtaining asample containing a pestivirus of interest or material thereof can beperformed according to standard methods as, for example, described inExample 2 or in Huist et al. (1994).

[0067] Therefore, in a preferred embodiment the present inventionrelates to a method for distinguishing pestivirus-infected animals fromanimals vaccinated with a specifically attenuated pestivirus accordingto the invention, comprising the following steps:

[0068] (1) Obtaining a sample from an animal of interest suspected ofpestivirus infection or a vaccinated animal;

[0069] (2) Determining the absence or presence of RNase activity of aglycoprotein E^(RNS) within said sample;

[0070] (3) Correlating the absence of RNase activity of glycoproteinE^(RNS) with a vaccinated animal and correlating the presence of saidactivity with a pestivirus infection of said animal.

[0071] The present invention provides pestiviruses inactivated in theirRNase activity residing in glycoprotein E^(RNS) by deletion and/ormutation. Such pestiviruses are easily detected because of thestructural consequences resulting from such deletions and/or mutations.The sequence difference of the E^(RNS) gene coding for the alteredglycoprotein E^(RNS) is detectable by sequencing techniques orPCR-techniques. As a result, the present invention provides in apreferred embodiment a method for distinguishing pestivirus-infectedanimals from animals vaccinated with a specifically attenuatedpestivirus according to the invention, comprising the following steps:

[0072] (1) Obtaining a sample from an animal of interest suspected ofpestivirus infection or a vaccinated animal;

[0073] (2) Identifying the nucleotide sequence of a pestivirus genome orprotein within said sample;

[0074] (3) Correlating the deletions and/or mutations of the E^(RNS)nucleotide sequence as present in the vaccine with a vaccinated animaland correlating the absence of said deletions and/or mutations with apestivirus infection of said animal.

[0075] Furthermore, the structural changes resulting from the alteredprotein sequence of the glycoprotein E^(RNS) of pestiviruses of theinvention can be detected by specific monoclonal or polyclonalantibodies, that do not recognize unaltered proteins.

[0076] Therefore, in a further embodiment, the present invention relatesto a method for distinguishing pestivirus-infected animals from animalsvaccinated with an attenuated pestivirus according to the invention,comprising the following steps:

[0077] (1) Obtaining a sample from an animal of interest suspected ofpestivirus infection or a vaccinated animal;

[0078] (2) Identifying a modified E^(RNS) glycoprotein of an attenuatedpestivirus by the specific binding of monoclonal or polyclonalantibodies to E^(RNS) glycoproteins present in said sample, saidglycoproteins being modified by a method according to the invention,whereby said monoclonal or polyclonal antibodies do not bind tounmodified E^(RNS) glycoproteins;

[0079] (3) Correlating the specific binding of said monoclonal orpolyclonal antibodies with a vaccinated animal and correlating theabsence of antibody binding to a pestivirus infection of said animalunder the proviso that the presence of pestiviral material in saidanimal and/or said sample is established otherwise.

[0080] Vice versa, it is also possible to detect the altered and therebystructurally labeled proteins by the absence of binding to specificmonoclonal or polyclonal antibodies that recognize unalteredglycoproteins E^(RNS) only, if the presence of pestiviruses can beestablished otherwise. In a preferred embodiment the present inventionrelates to a method for distinguishing pestivirus-infected animals fromanimals vaccinated with an attenuated pestivirus according to theinvention, comprising the following steps:

[0081] (1) Obtaining a sample from an animal of interest suspected ofpestivirus infection or a vaccinated animal;

[0082] (2) Identifying an unmodified E^(RNS) glycoprotein of apestivirus by the specific binding of monoclonal or polyclonalantibodies to E^(RNS) glycoproteins present in said sample, saidglycoproteins not being modified by a method according to the invention,whereby said monoclonal or polyclonal antibodies do not bind to modifiedE^(RNS) glycoproteins;

[0083] (3) Correlating the specific binding of said monoclonal orpolyclonal antibodies with a pestivirus infection in said animal andcorrelating the absence of antibody binding to an vaccinated animalunder the proviso that the presence of pestiviral material in saidanimal and/or said sample is established otherwise.

[0084] Of course, the structural modification and absence of the RNaseactivity in the labeled viruses of the invention will result indifferent immune responses in animals when compared to the responsesresulting from unlabeled pestivirus infections. The pestiviruses of theinvention elicit a different and distinct immune response, cellular aswell as humoral, that differs from unmodified and possibly pathogenicimmune responses. For example, glycoproteins E^(RNS) according to theinvention will result in polyclonal antibodies that are different intheir binding specificity when compared to polyclonal antibodiesresulting from unmodified glycoproteins. This difference in bindingspecificity provides a label for distinguishing animals vaccinated withpestiviruses from the invention from pestivirus field infected animals.Tests for screening sera for specific polyclonal antibodies that eitherbind to a wildtype epitope or a marker deletion mutation of that epitopefor the purpose of differentiating infected and vaccinated animals havebeen described, for example for pseudorabies-infected and vaccinatedpigs (Kit et al., 1991).

[0085] In a preferred embodiment the present invention relates to amethod for distinguishing pestivirus-infected animals from animalsvaccinated with an attenuated pestivirus according to the invention,comprising the following steps:

[0086] (1) Obtaining a sample of polyclonal antibodies from an animal ofinterest suspected of pestivirus infection or a vaccinated animal;

[0087] (2) Identifying any specific binding of said polyclonalantibodies to unmodified glycoprotein E^(RNS) or glycoprotein E^(RNS)RNs as modified according to the invention.

[0088] (3) Correlating the binding of said polyclonal antibodies tounmodified glycoprotein E^(RNS) with a pestivirus infection andcorrelating the binding of said polyclonal antibodies to glycoproteinE^(RNS) as modified according to the invention with a vaccinated.

References

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[0090] 2. Becher, P., König, M., Paton, D. J., Thiel, H. J., 1995,Further characterization of border disease virus isolates: evidence forthe presence of more than three species within the genus pesivirus.Virology 209 (1), 200-206.

[0091] 3. Donis, R. O., Corapi, W., and Dubovi, E. J. 1988. Neutralizingmonoclonal antibodies to bovine viral diarrhea virus bind to the 56K to58K glycoprotein. J. Gen. Virol. 69: 77-86.

[0092] 4. Fuerst T. R. et al. 1986. Eukaryotic transient expressionsystem based on recombinant vaccinia virus that synthesizesbacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. 83: 8122-8126.

[0093] 5. Hulst, M. M., Himes, G., Newbigin, E., Moormann, R. J. M.1994. Glycoprotein E2 of classical swine fever virus: expression ininsect cells and identification as a ribonuclease. Virology 200:558-565.

[0094] 6. Hulst, M. M., F. E. Panoto, A. Hooekmann, H. G. P. vanGennip., and Moormann, R. J. M. 1998. Inactivation of the RNase activityof glycoprotein E^(ms) of classical swine fever virus results in acytopathogenic virus. J. Virol 72: 151-157.

[0095] 7. Kit, M. and S. Kit. 1991. Sensitive glycoprotein gill blockingELISA to distinguish between pseudorabies (Aujeszky's disease)-infectedand vaccinated pigs. Veterinary Microbiology 28:141-155.

[0096] 8. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapidand efficient site-specific mutagenesis without phenotypic selection.Methods Enzymol. 154:367-392.

[0097] 9. König, Matthias, 1994, Virus der klassischen Schweinepest:Untersuchungen zur Pathogenese und zur Induktion einer protektivenImmunantwort. Dissertation, Tierärztliche Hochschule Hannover, Germany.

[0098] 10. Meyers, G., Rümenapf, T. and Thiel, H. -J. 1989. Molecularcloning and nucleotide sequence of the genome of hog choleravirus.Virology 171: 555-567.

[0099] 11. Meyers, G., Tautz, N., Becher, P., Thiel, H. -J., & Kümmerer,B. M. 1996b. Recovery of cytopathogenic and noncytopathogenic bovineviral diarrhea viruses from cDNA constructs. J. Virol, 70: 8606-8613.

[0100] 12. Meyers, G., Thiel, H. -J., and Rümenapf, T. 1996a. Classicalswine fever virus: Recovery of infectious viruses from cDNA constructsand generation of recombinant cytopathogenic swine fever virus. J.Virol. 67:7088-709526.

[0101] 13. Moennig, V. and Plagemann, J. 1992. The pestiviruses. Adv.Virus Res. 41: 53-91.

[0102] 14. Paton, D. J., Lowings, J. P., Barrett, A. D. 1992. Epitopemapping of the gp53 envelope protein of bovine viral diarrhea virus.Virology 190: 763-772.

[0103] 15. Pellerin, C. et. al. Identification of a new group of bovineviral diarrhea virus strains associated with severe outbreaks and highmortalities, Virology 203, 1994:260-268.

[0104] 16. Rice, C. M. 1996. The pestiviruses. In Fields Virology, eds.Fields, B. N., Knipe, D. M., & Howley, P. M. (Lippincott-Raven,Philadelphia), pp. 931-959.

[0105] 17. Rümenapf, T., Unger, G., Strauss, J. H., and Thiel, H. -J.1993. Processing of the evelope glycoproteins of pestiviruses. J. Virol.67: 3288-3294

[0106] 18. Schneider, R., G. Unger, R. Stark, E. Schneider-Scheizer, andH. -J. Thiel. 1993. Identification of a structural glycoprotein of anRNA virus as a ribonuclease. Science 261: 1169-1171.

[0107] 19. Thiel, H. -J., Plagemann, G. W., & Moennig, V. 1996. Thepestiviruses. In Fields Virology, eds. Fields, B. N., Knipe, D. M., &Howley, P. M. (Lippincott-Raven, Philadelphia), pp.1059-1073.

[0108] 20. Thiel, H. -J., Stark, R., Weiland, E., Rümenapf, T. & Meyers,G. 1991. Hog cholera virus: molecular composition of virions from apestivirus. J. Virol. 65: 4705-4712.31.

[0109] 21. van Rijn, P. A., van Gennip, H. G., de Meijer, E. J.,Moormann, R. J. 1993. Epitope mapping of envelope glycoprotein E1 of hogcholera virus strain Brescia. J. Gen. Virol. 74: 2053-2060.

[0110] 22. Weiland, E., Thiel, H. -J., Hess, G., and Weiland, F. (1989).Development of monoclonal neutralizing antibodies agaist bovine viraldiarrhea virus after pretreatment of mice with normal bovine cells andcyclophosphamide. J. Virol. Methods 24: 237-244.

[0111] 23. Weiland, E., Stark, R., Haas, B., Rümenapf, T., Meyers, G.and Thiel, H. -J. (1990). Pestivirus glycoprotein which inducesneutralizing antibodies forms part of a disulfide-linked heterodimer. J.Virology 64, 3563-3569.

[0112] 24. Weiland, E., Ahl, R., Stark, R., Weiland, F. and Thiel, H.-J. (1992). A second envelope glycoprotein mediates neutralization of apestivirus, hog cholera virus. J. Virology 66, 3677-3682.

[0113] 25. Windisch, J. M., Schneider, R., Stark, R., Weiland, E.,Meyers, G., and Thiel, H. -J. 1996. RNase of classical swine fevervirus: biochemical characterization and inhibition by virus-neutralizingmonoclonal antibodies. J. Virol. 70: 352-358

EXAMPLES Example 1

[0114] Generation of RNase-negative pestivirus mutants

[0115] Starting with the full length cDNA clones pA/CSFV (Meyers et al.,1996a) or pA/BVDV (Meyers et al., 1996b), from which infectious cRNA canbe obtained by in vitro transcription, subclones were generated. ForCSFV, a Xhol/Sspl fragment of pA/CSFV was cloned into pBluescript SK+,cut with Xhol and Smal. For BVDV, a Xhol/BgIII fragment from pA/BVDV wascloned into plasmid pCITE-2C, cut whit the same enzymes. Single strandedplasmid DNA was produced from these constructs according to the methodof Kunkel (Kunkel et al., 1987) using E. coli CJ 236 cells (BioRad) andthe VCMS single strand phage (Stratagene). The single stranded DNA wasconverted to double strands using the ‘Phagemid in vitro MutagenesisKit’ (BioRad). Some of the synthetic oligonucleotides which were used asprimers for generating the desired pestivirus mutants are listed belowin an exemplary fashion: C-297-L: AGGAGCTTACTTGGGATCTG C-346-L:GGAACAAACTTGGATGGTGT C-297-K: ACAGGAGCTTAAAAGGGATCTGGC C-346-K:ATGGAACAAAAAGGGATGGTGTAA C-346-d: GAATGGAACAAAGGATGGTGTAAC B-346-d:CATGAATGGAACAAAGGTTGGTGCAACTGG

[0116] The double stranded plasmid DNA was used for transformation of E.coli XL1-Blue cells (Stratagene). Bacterial colonies harboring plasmidswere isolated via ampicillin selection. Plasmid DNA was prepared andfurther analyzed by nucleotide sequencing using the T7 polymerasesequencing kit (Pharmacia). Plasmids containing the desired mutationsand no second site changes were used for the construction of full lengthcDNA clones. In the case of CSFV, a Xhol/Ndel fragment from themutagenized plasmid was inserted together with a Ndel/BgIII fragmentderived from plasmid 578 (PCITE 2A, containing the Xhol/BgIII fragmentform pA/CSFV) into pA/CSFV cut with Xhol and BgIII. To obtain the BVDVCP7 mutant, a Xhol/BgIII fragment containing the deletion was insertedinto pA/BVDV cut with Xhol and Ncol together with a BgIII/Ncol fragmentisolated from pA/BVDV/lns-. From construct pA/BVDV/lns- a cRNA wastranscribed that gives rise to a noncytopathogenic BVDV upontransfection in suitable cells (Meyers et al., 1996b).

[0117] The different full length clones were amplified, and the plasmidsisolated. The presence of the desired mutations was proven by DNAsequencing. After linearization with Srfl (CSFV full length clones) orSmal (BVDV full length clones) cRNA was transcribed as describedpreviously (Meyers et al., 1996ab). RNA was purified by gel filtrationand phenol/chloroform extraction and used for transfection of porcinekidney (PK15) cells or bovine kidney (MDBK clone B2) cells (CSFV or BVDVconstructs, respectively). The transfections were analyzed byimmunofluorescence with virus specific antisera. In cases where thedesired mutants could be recovered (immunofluorescence positive) theviruses were amplified by passage on the same cell lines used for thetransfection experiments. Further analysis of the CSFV mutants includeddetermination of one step growth curves and characterization of viralRNA by Northern blot with virus specific cDNA probes as well as reversetranscription polymerase chain reaction (RT-PCR) and subsequentsequencing of the PCR fragments to verify the presence of the desiredmutations in the viral genome. In all cases the presence of the desiredmutation was proven. All of the recovered viruses grew equally well andproduced similar amounts of RNA just as the virus resulting from theplasmid displaying the wild type sequence.

[0118] The viability of the BVDV mutant was shown by transfection of therespective cRNA and splitting of the cells 3 days thereafter. Part ofthe cells was seeded into a 3.5 cm diameter dish, fixed withacetone/methanol at the day thereafter and analyzed byimmunofluorescence with a mixture of BVDV-specific monoclonal antibodies(Weiland et al., 1989). All cells were found positive whereas a controlof cells transfected with noninfectious RNA showed no signal. From apart of the cells transfected with the respective cRNA, an extract wasproduced by one cycle of freezing and thawing. Fresh cells were infectedwith this cell extract and proved to be BVDV positive by BVDV specificimmunofluorescence 3 days post infection.

[0119] Table 1 summarizes the different changes introduced into theconserved sequences of E^(RNS) representing the putative active site ofthe RNase which are encoded by the indicated virus mutants TABLE 1 RNaseViability Name Sequence in RNase motif activity of mutant pA/CSFV...SLHGIWPEKIC... ...RHEWNKHGWCNW.. + + C-297-L ...SLLGIWPEKIC......RHEWNKHGWCNW.. − + C-346-L ...SLHGIWPEKIC... ...RHEWNKLGWCNW.. − +C-297-L/346-L ...SLLGIWPEKIC... ...RHEWNKLGWCNW.. − + C-297-K...SLKGIWPEKIC... ...RHEWNKHGWCNW.. − + C-346-K ...SLHGIWPEKIC......RHEWNKKGWCNW.. − + C-297-d ...SL GIWPEKIC... ...RHEWNKHGWCNW.. − −C-346-d ...SLHGIWPEKIC... ...RHEWNK GWCNW.. − + C-296/7/8-d...S   IWPEKIC... ...RHEWNKHGWCNW.. − − C-345/6/7-d ...SLHGIWPEKIC......RHEWN   WCNW.. − − C-345/6-d ...SLHGIWPEKIC... ...RHEWN  GWCNW.. − −C-346/7-d ...SLHGIWPEKIC... ...RHEWNK  WCNW.. − − C-342-d...SLHGIWPEKIC... ...RH WNKHGWCNW.. − − C-342/6-d ...SLHGIWPEKIC......RH WNK GWCNW.. − − C-301-d ...SLHGIW EKIC... ...RHEWNKHGWCNW.. − −C-295-S/G ...GLHGIWPEKIC... ...RHEWNKHGWCNW.. − + C-300-W/G...SLHGIGPEKIC... ...RHEWNKHGWCNW.. − + C-302-E/A ...SLHGIWPAKIC......RHEWNKHGWCNW.. − − C-305-C/G ...SLHGIWPEKIG... ...RHEWNKHGWCNW.. − −C-300-W/G-302-E/A ...SLHGIGPAKIC... ...RHEWNKHGWCNW.. − − C-340-R/G...SLHGIWPEKIC... ...GHEWNKHGWCNW.. − − C-343-W/G ...SLHGIWPEKIC......RHEGNKHGWCNW.. − − C-345-K/A ...SLHGIWPEKIC... ...RHEWNAHGWCNW.. − −C-297-K/346-K ...SLKGIWPEKIC... ...RHEWNKKGWCNW.. − + C-297-K/346-L...SLKGIWPEKIC... ...RHEWNKKGWCNW.. − + pA/BVDV ...SLHGIWPEKIC......RHEWNKHGWCNW.. + + B-346-d ...SLHGIWPEKIC... ...RHEWNK GWCNW.. − + #determined as described below.

Example 2

[0120] Effect of different mutations on RNase activity of E^(RNS)

[0121] To test the effect of the different mutations on the RNaseactivity of E^(RNS) appropriate cells were infected with the mutantviruses. For CSFV, the infection was carried out with a multiplicity ofinfection (m.o.i.) of 0.01. Infection with wild type virus served as apositive control whereas noninfected cells were used as a negativecontrol. At 48 h post infection, cells were washed twice with phosphatebuffered saline and lysed in 0.4 ml of lysis buffer (20 mM Tris/HCI; 100mM NaCl, 1 mM EDTA, 2 mg/ml bovine serum albumin; 1% Triton X100; 0.1%deoxycholic acid; 0.1% sodium dodecyl sulfate). The lysate was giveninto 1.5 ml reaction tubes, sonified (Branson sonifier B12, 120 Watt, 20s in a cup horn water bath), cleared by centrifugation (5 min, 14,000rpm, Eppendorf Centrifuge, 4° C.) and the supernatant subjected toultracentrifugation (Beckmann table top ultracentrifuge, 60 min at 4° C.and 45,000 rpm in a TLA 45 rotor). Determination of RNase activity wasdone in a total volume of 200 μl containing 5 or 50 μl of supernatant ofthe second centrifugation step and 80 μg of Poly(rU)(Pharmacia) inRNase-assay buffer (40 mM Tris-acetate (pH 6.5), 0.5 mM EDTA, 5 mMdithiothreitol (DTT)). After incubation of the reaction mixture at 37°C. for 1 hour 200 μl of 1.2 M perchloric acid, 20 mM LaSO₄ was added.After 15 min incubation on ice the mixture was centrifugated for 15 minat 4° C. and 14,000 rpm in an Eppendorf centrifuge. To the supernatant 3volumes of water were added and an aliquot of the mixture was analyzedby measuring the optical density at 260 nm using an Ultrospec 3000spectrophotometer (Pharmacia). In all cases, the mutations introducedinto the E^(ms) gene completely abrogated RNase activity (Table 1).

[0122] For the BVDV mutant RNase activity was tested with materialobtained after RNA transfection without passage of the recoveredviruses. Cells transfected with the appropriate RNA were split 72 h posttransfection and seeded in two dishes. 24 h later, from one dish, cellextracts were prepared and analyzed for RNase activity as describedabove. To prove infection, the cells of the second dish were analyzed byimmunofluorescence with BVDV specific monoclonal antibodies (Weiland etal., 1989) and found 100% positive. Transfection was carried out withRNA transcribed from pA/BVDV/lns- and from pA/B-346-d, the plasmidequivalent to pA/BVDV/lns- but containing the deletion of the codonequivalent to the codon 346 in the CSFV Alfort genome. NontransfectedMDBK cells served as a negative control. TABLE 2A Determination of RNaseactivity of different viruses Alfort C-WT C-297-L C-346-L C-346-dC-346-d/Rs control OD₂₆₀ 2.4 2.3 1.1 1.1 1.1 2.3 1.1  Alfort C-WTC-297-L C-346-L C-297-K C-346-K C-297-L/346-L OD₂₆₀ 2.09 2.16 0.715 0.770.79 0.766 0.77 C-297-K/346-L C-297-K/346-K C-346-d Control OD₂₆₀ 0.7250.835 0.8 0.84 #obtained after quantification of E^(RNS) by radioactivelabeling, immunoprecipitation and analysis of radioactivity with aphosphorimager. Moreover, reduction of the E^(ms) concentration in theassay down to only one tenth of the usual amount did not change theresulting OD values considerably, indicating that with the chosenconditions the assay was saturated with E^(ms).

[0123] TABLE 2B Description of table 2B B-WT B-346-d control OD₂₆₀ 2.51.1 1.1 # analyzed 24 h later for RNase activity. Infection of the cellswas proven by immunofluorescence # analysis as described in the text. #extract from noninfected MDBK cells.

Example 3

[0124] Pathogenicity of CSFV after RNase inactivation

[0125] To test, whether the destruction of the RNase activity influencesthe pathogenicity of pestiviruses in their natural host, animalexperiments were conducted with mutant V(pA/C-346-d) (C346-d in tables).Virus recovered from the CSFV full length clone without mutation(V(pA/CSFV)) served as a positive control (C-WT in tables). For eachmutant three piglets (breed: German landrace; about 25 kg body weight)were used. The infection dose was 1×10⁵ TCID₅₀ per animal; two thirds ofthe inoculate was administered intranasally (one third in each nostril),one third intramuscularly. The two groups were housed in separateisolation units. Blood was taken from the animals two times beforeinfection and on days 3, 5, 7, 10, 12 and 14. In addition, temperaturewas recorded daily (FIG. 2). The animals infected with the wild typevirus showed typical symptoms of classical swine fever like fever,ataxia, anorexia, diarrhea, central nervous disorders, hemorrhages inthe skin (Table 3a). Virus could be recovered form the blood on days 3(animal #68) and on days 5, 7, 10, 14 (animals #68, #78, #121) (Table3b) The animals were killed in a moribund stage at day 14 postinfection. At this time, no virus neutralizing antibodies could bedetected. In contrast, the animals infected with the mutant did notdevelop clinical symptoms (Table 3a). The temperature stayed normal(FIG. 2) over the whole experimental period and the animals neverstopped taking up food. At no time virus could be recovered from theblood. Nevertheless, the animals were clearly infected and the virusmost likely replicated since all animals developed neutralizingantibodies (Table 3c). TABLE 3a Clinical signs after test infection:Animal experiment 1 clinical signs hemor- moribund at Hemorrhages Anim.diar- CNS ano- rhages day of in organs at No.: infected with fever rheadisorders rexia in skin apathia euthanasia necropsy #68C-WT + + + + + + + + #78 C-WT + + + + + + + + #121 C-WT + + + + + + + +#70 C-346-d − − − − − − − n.a. #72 C-346-d − − − − − − − n.a. #74C-345-d − − − − − − − n.a.

[0126] TABLE 3b Blood cell viremia after test infection Animalexperiment 1 Animal infected viremia at days post infection number with3 5 7 10 14 #68 C-WT + + + + + #78 C-WT − + + + + #121 C-WT − + + + +#70 C-346-d − − − − − #72 C-346-d − − − − − #74 C-346-d − − − − −

[0127] TABLE 3c Development of CSFV specific serum neutralization titerdays p.i. −3 0 17 25 69 76 79 87 pig #70 — — 1:18  1:162 1:162 1:1621:486 1:1458 pig #72 — — 1:18 1:54 1:486  1:1458  1:1458 1:4374 pig #74— — 1:6  1:54 1:162 1:162 1:486 1:1458 #infection the animals werechallenged with 2 × 10⁵ TCID₅₀ of CSFV strain Eystrup. The table givesthe highest serum dilution resulting in complete neutralization of inputvirus.

Example 4

[0128] Induction of protective immunity by infection with RNase negativevirus

[0129] To analyze whether the infection with the mutant virus had led toa protective immunity, a challenge experiment was conducted about 9weeks after the infection with the CSFV mutant using a highly pathogenicheterologous CSFV strain (strain Eystrup, originated from Behring).2×10⁵ TCID₅₀ of virus was used for the infection. This amount of viruswas found to be sufficient to induce lethal disease in severalpreceeding experiments (König, 1994). However, the animals previouslyinfected with the CSFV RNase mutant did not show symptoms of diseaseafter challenge infection. Neither fever (FIG. 3) nor viremia could bedetected but an increase in neutralizing antibodies indicated productiveinfection and replication of the challenge virus.

Example 5

[0130] Confirmation of attenuation principle

[0131] To show, that the observed attenuation of the mutant virus isindeed due to the deletion of the histidine at position 346 of thepolyprotein and not a consequence of an unidentified second sitemutation, the wild type sequence was restored by exchange of a 1.6 kbXhol/Ndel fragment of the full length clone pA/C-346-d against thecorresponding fragment of pA/CSFV displaying the wild type sequence. Thefragment excised from pA/C-346-d was analyzed by nucleotide sequencingfor mutations. Except for the deletion of the triplet coding forhistidine 346 of the polyprotein, no difference with regard to the wildtype sequence was found. From the cDNA construct with the rescuedmutant, virus V(pA/C-346-d/Rs) could be recovered that grew equally wellas wild type virus and showed equivalent RNase activity (Table 2A).

[0132] In a second animal experiment, the rescued virus was used forinfection of pigs. As a control, the deletion mutant was used. Again,two groups consisting of three animals were used. As the animals wereyounger (German landrace, about 20 kg) than those in the firstexperiment, 5×10⁴ TCID₅₀ of virus were used for infection this time.Again, the animals infected with the mutant showed no clinical signs(Table 5, FIG. 4). Only one animal had fever for one day. Nevertheless,these animals developed neutralizing antibodies and were protectedagainst a lethal CSFV challenge. Challenge was again performed byinfection with 2×10⁵ TCID₅₀ of challenge strain Eystrup. The animals didnot show clinical signs after challenge and the temperature stayednormal (FIG. 5). In contrast to the pigs infected with the deletionmutant, the animals inoculated with the rescued wild type virusdeveloped fatal classical swine fever. One animal had to be killed 11days after infection, the other two 3 days later. All animals showedtypical symptoms of classical swine fever, i.e. fever, diarrhea,annorexia, and pathological signs like hemorrhages in different organsincluding the kidney. TABLE 5a Clinical signs after test infectionAnimal experiment 2 clinical signs hemor- moribund at hemorrhages Anim.CNS rhages in day of in organs at No.: infected with fever diarrheadisorders anorexia skin apathia euthanasia necropsy #43 C-346-d +* − − −− − − n.a. #47 C-346-d − − − − − − − n.a. #87 C-346-d − − − − − − − n.a.#27 C-346-d/RS + + + + − + + + #28 C-346-d/RS + + + + − + + + #30C-346-d/RS + + + + − + + + #was performed.

[0133] TABLE 5b Diagnostic RNAse test with viruses recovered frominfected animals during viremia animal #3 animal #5 animal #27 animal#28 animal #30 Con- Alfort C-297-K C-297-K C-346-d/RS C-346-d/RSC-346-d/RS trol OD₂₆₀ 1.84 0.60 0.56 1.84 1.93 1.94 0.49

[0134] Viruses recovered form the blood of animals 3 and 5 at day 5 postinfection and of animals 27, 28 and 30 of animal experiment #2(described in example 5) at day 7 post infection were propagated intissue culture, titrated and tested for RNase activity as describedabove. Non-infected PK15 cells and cells (control) infected with wildtype CSFV (Alfort) served as controls. Animals 3 and 5 had been infectedwith mutant C-297-K, whereas animals 27, 28 and 30 had been infectedwith mutant C-346-d/RS, as indicated in the table.

Example 6

[0135] Effects of double mutation within E^(RNS)

[0136] To test the effects of a double mutation within E^(RNS) on theability of the respective virus to replicate in its natural host and onpathogenicity, an animal experiment was conducted with mutantV(pA/C-297-L/346-L). Virus recovered from the CSFV full length clonewithout mutation (V(pA/CSFV) served as a positive control. For eachmutant three piglets (breed: German land race; about 25 kg body weight)were used. The infection dose was 1×10⁵ TCID₅₀ per animal; two thirds ofthe inoculate was administered intra-nasally (one third in eachnostril), one third intramuscularly. Blood was taken from the animalsbefore infection (day 0) and on days 5, 8, 12 and 20. In addition,temperature was recorded daily (FIG. 6). The animals infected with thedouble mutant did not develop any clinical symptoms, and the animalsnever stopped taking up food. The animals showed no fever over the wholeexperimental period (animals 45/2 and 45/3) except animal 45/1 on day 8,probably due to bacterial infection caused by injury of the right hindleg. After treatment of this animal with an antibiotic on day 10,temperature returned to normal values within one day (FIG. 6). For allanimals virus was recovered from the blood on day 5 whereas no viremiawas detected at later time points (Table 6a). All animals developedneutralizing antibodies (Table 6b). For animal 45/1 the neutralizationtiter was again determined about 4.5 months p.i. and was found to be1:4374. Thus, the infection with the double mutant resulted in longlasting immunological memory. TABLE 6a Test for viremia Days p.i. 5 8 12Pig 45/I + − − Pig 45/II + − − Pig 45/III + − −

[0137] TABLE 6b Neutralization titers Animal day 0 day 20 p.i. 45/1 −1:128 45/2 − 1:256 45/3 − 1:256

Example 7

[0138] Immunogenicity and attenuation principle of the BVDV virus“B-346-d”

[0139] This experiment was designed to investigate the attenuationprinciple as well as the immunogenicity of the BVDV virus ,B-346-d'recovered from pA/B-346-d by comparing it with the ,B-WT' virusrecovered from pA/BVDV/Ins-. The virus ,B-346-d' is of course mutated inoriginal BVDV position 349 but named “B-346” to indicate the positionrelative to the CSFV Alfort position 346 of FIG. 1.

[0140] Three groups of BVDV seronegative animals of 3-6 months of agewere selected. Groups 1 and 2 comprised 5 animals each while group 3comprised 3 animals. Animals of group 1 and 2 were infected byadministration of 2×10⁶ TCID₅₀ of B-346-d (group 1) or B-WT (group 2) ina volume of 5 ml per route. Animals were infected intra-muscularly(gluteal muscle), intranasally and subcutaneously (over scapula). Over aperiod of 14 days after infection, viremia in both groups was monitoredthrough parameters like blood cell viremia and virus shedding in nasalswabs. In addition, clinical parameters like rectal temperatures, whiteblood cell counts and general health parameters were monitored.

[0141] The protective immunity against an infection with anantigenetically heterologous and virulent BVDV-isolate (#13) wasinvestigated by challenge infection 77 days after infection of theanimals of group 1 with B-346-d. Animals of group 3 served as challengecontrol and were infected according to the procedure for the animals ofgroup 1 with the virulent BVDV-isolate. The BVDV virus (#13) belongs toa different antigenetic group (type II), whereas the B-346-d virusbelongs to the antigenetic group (type I) according to theclassification described by (Pellerin, C. et. al., 1994). Animals ofgroup 1 and 3 got infected by administration of 2×10⁶ TCID₅₀ of BVDVisolate (#13) in a volume of 5 ml per route. Animals were infected viathe intra-muscular (gluteal muscle), intra-nasal and subcutaneous route(over Scapula). Over a period of 14 days after infection viremia in bothgroups was monitored by parameters like blood cell viremia and virusshedding in nasal swabs. In addition, clinical parameters like rectaltemperatures, white blood cell counts and general health parameters weremonitored.

[0142] After infection with B-346-d animals did not show any typicalclinical symptoms of a BVDV infection such as rectal temperatureincrease (Table 7a), or any respiratory clinical symptomes (not shown).

[0143] The reduced blood cell viremia (Table 7b) and virus shedding innasal swabs (Table 7c) did clearly indicate an attenuation of B-346-dcompared to B-WT.

[0144] The virulent BVDV isolate #13 did induce in the animals of group3 a strong viremia with typical signs of a BVDV infection, like rectaltemperature increase over a period of several days (Table 7d), strongleucopenia (Table 7e), extended blood cell viremia (Table 7f) and virusshedding in nasal swab fluid (Table 7g). In contrast, animals of group1, which had been vaccinated by infection with B-346-d, did show almostno clinical symptoms typical for a BVDV infection after the challengeinfection with the virulent BVDV isolate #13. There was no significantincrease in rectal temperatures after infection (Table 7d). The observedleucopenia was very marginal with regard to magnitude and duration(Table 7e). No BVDV could be isolated from the blood (Table 7f) and foronly one animal virus shedding in nasal swab exudate could be detected(Table 7g).

[0145] Therefore, infection with B-346-d induces a strong immunity whichclearly reduces clinical signs, virus shedding and blood cell viremiaafter challenge infection with a heterologous BVDV isolate. TABLE 7aMean rectal temperatures in group 1 (B-346-d) and 2 (B-WT) Day of study:0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Group 1 38,8 39,1 39,0 38,7 38,8 38,738,7 38,5 38,7 38,5 38,5 38,5 38,4 38,9 38,7 Group 2 38,8 39,0 38,9 38,638,6 38,7 38,6 38,4 39,1 38,4 38,7 38,6 38,7 38,6 38,6

[0146] FIG. 7b Blood cell viremia of groups 1 and 2 First day nasalFinal day nasal Recorded duration of Mean duration of Group Animalshedding recorded shedding recorded nasal shedding (days) group (days) 11 6 6 1 1,4 2 4 6 2 3 5 5 1 4 — — 0 5 6 9 3 2 6 4 8 5 4,4 7 4 7 4 8 4 74 9 4 7 4 10  4 8 5

[0147] EDTA blood was sampled daily up to day 10 post infection withB-346-d and B-WT, respectively. 2.0 ml of blood were added to each of 3cultures of calf testis (Cte) cells with medium containing heparin (1unit/ml to prevent clotting). After overnight incubation inoculum/mediumwas replaced with fresh medium without heparin. After incubation for 4to 6 days, BVDV infected cells were detected by immunefluorescence witha polyclonal serum specific for BVDV. Negative cultures were frozen andsubsequently thawed. 0.2 ml thereof were passed to a second passage onCte cells to confirm the absence of BVDV. TABLE 7c Virus shedding innasal fluid: First day Number of nasal Final day nasal days virus Meannumber of shedding shedding detected in days detected Group Animalrecorded recorded exudate virus per group 1 1 4 8 4 2,6 2 6 6 1 3 4 4 14 5 7 3 5 3 6 4 2 6 6 8 3 3,6 7 5 7 3 8 5 8 4 9 5 6 2 10  3 9 6

[0148] Nasal exudate was centrifuged (1000 g) to remove gross debris andcontaminants. Supernatant fluid was removed and 0,2 ml were seeded toeach of three cell cultures. After overnight incubation theinoculum/medium was replaced with 2 ml of fresh medium. After incubationfor 4-6 days, BVDV infected cells were detected by immunofluorescencewith a polyclonal serum specific for BVDV. TABLE 7d Mean rectaltemperatures in group 1 and 3 Day of study: −2 −1 0 1 2 3 4 5 6 7 8 9 1012 14 Group 1 38,4 38,6 38,5 38,5 38,6 38,4 38,4 38,4 38,3 38,4 38,438,4 38,4 38,4 38,5 Group 3 38,8 39,1 38,8 39,1 39,4 39,7 40,2 40,2 40,441,3 40,2 40,1 40,2 40,8 40,4

[0149] TABLE 7e Mean white blood cell counts Day of study: −2 −1 0 1 2 34 5 6 7 8 9 10 12 14 Group 1 11,9 11,9 11,3 10,8 9,2 8,2 8,9 9,9 11,211,6 11,6 10,6 10,8 10,8 9,4 Group 3 11,7 15,8 13,8 11,1 7,7 9,8 7,4 6,8 7,5  8,7  7,0  8,1  6,2  6,4 6,2

[0150] EDTA blood cell samples were taken daily from day −2 to 14 postchallenge from each animal in both groups. Counts of white blood cellsin EDTA blood samples were determined using a Sysmex Micro-Cell CounterF800. TABLE 7f BVDV isolated from blood samples First Final Recorded dayvirus day virus duration of Mean detected in detected in virus in bloodduration Group Animal blood blood (days) (days) 1 1 — — 0 0 2 — — 0 3 —— 0 4 — — 0 5 — — 0 3 11 3 10 8 9,7 12 3 14 12 13 3  9 9

[0151] EDTA blood was sampled daily up to day 10 post challenge. 0.2 mlof blood were added to each of 3 cultures of calf testis (Cte) cellswith medium containing heparin (1 unit/ml to prevent clotting). Afterovernight incubation inoculum/medium was replaced with fresh mediumwithout heparin. After incubation for 4 to 6 days cells BVDV infectedcells were detected by immunefluoreszence with a polyclonal serumspecific for BVDV.

[0152] Negative cultures were frozen and subsequently thawed. 0.2 mlthereof were passed to a second passage on Cte cells to confirm theabsence of BVDV. TABLE 7g Virus shedding in nasal fluid First day Finalday Recorded Mean nasal nasal duration duration shedding shedding ofnasal (days, Group Animal recorded recorded shedding (days) per group) 11 3 4 2 0,8 2 — — 0 3 — — 0 4 — — 0 5 4 5 2 3 11 3 14 12 10 12 3 14 1213 3 8 6

[0153] Nasal exudate was centrifuged (1000 g) to remove gross debris andcontaminants. Supernatant fluid was removed and 0,2 ml thereof wereseeded to each of three cell cultures. After overnight incubation theinoculum/medium was replaced with 2 ml of fresh medium. After incubationfor 4-6 days BVDV infected cells were detected by immunefluorescencewith a polyclonal serum specific for BVDV.

Example 8

[0154] Discrimination between C-346-d and CSFV without deletion of thehistidine codon 346 by RT-PCR

[0155] The RNA sequence coding for the conserved RNase motif in CSFVglycoprotein E^(RNS) highly conserved. Among all known CSFV sequences nonucleotide exchanges were found in the region corresponding to residues1387 to 1416 of the published sequence of the CSFV Alfort strain (Meyerset al., 1987). Thus, oligonucleotide primers derived from this conservedregion of the genome can be used in an RT-PCR assay for detection of allCSFV isolates (see FIG. 7). In consequence, the absence of the tripletcoding for histidine 346 (nucleotides 1399-1401) could be detected by anRT-PCR assay with an appropriately designed primer. Differentoligonucleotides covering the conserved region were synthesized thateither contained the histidine codon or not. These oligonucleotidesserved as upstream primers in RT-PCR reactions with oligonucleotideE^(RNS)-Stop as downstream primer. RNA purified from tissue culturecells infected with C-346-d, C-WT, C-346-L or C-346-K, respectively,were used as templates. Reverse transcription of 2 μg heat denatured RNA(2 min 92° C., 5 min on ice in 11.5 μl of water in the presence of 30pMol reverse primer) was done after addition of 8 μl RT mix (125 mMTris/HCI pH 8.3, 182.5 mM KCI, 7.5 mM MgCl₂, 25 mM dithiothreitol, 1.25mM of each dATP, dTTP, dCTP, dGTP), 15 U of RNAguard (Pharmacia,Freiburg, Germany) and 50 U of Superscript (Life Technologies/BRL,Eggenstein, Germany) for 45 min at 37° C. After finishing reversetranscription, the tubes were placed on ice and 30 μl of PCR mix (8.3 mMTris/HCI, pH8.3; 33.3 mM KCI; 2.2 mM MgCl₂; 0.42 mM of each dATP, dTTP,dCTP, dGTP; 0.17% TritonX100; 0.03% bovine serum albumine; 5 U of Taqpolymerase (Appligene, Heidelberg, Germany) and 16.7% DMSO) were added.When primer Ol H+3 was used, the reaction mix for amplificationcontained no DMSO. Amplification was carried out in 36 cycles (30 sec94° C.; 30 sec 57° C.; 45 sec 74° C.) 1 μl of amplification reaction wasloaded on a 1% agarose gel, the amplified products were separated byelectrophoresis, and stained with ethidium bromide. As demonstrated inFIG. 7, primer pair Ol H−3/Ol E^(ms)Stop allowed to specifically amplifya band derived from RNA containing the deletion of codon 346 whereaswith the other two primer combinations products containing codon 346were amplified and no band was observed when the RNA with the deletionof this codon was used as a template. Primers for RT-PCR:      upstream:        OI H−3: TGGAACAAAGGATGGTGT         OI H+2: TGGAACAAACATGGATGG        OI H+3: GAATGGAACAAACATGGA      downstream:         OIE^(ms)Stop: GGAATTCTCAGGCATAGGCACCAAACCAGG

FIGURE LEGENDS

[0156]FIG. 1: The first 495 amino acids as expressed by the Alfortstrain of CSFV

[0157] The sequence listing shows the first 495 amino acids as expressedby the Alfort strain of CSFV (Meyers et al., 1989). One monomer of theglycoprotein E^(RNS) of said strain corresponds to the amino acids 268to 494 as described by Rümenapf et al. (1993). Residues 295 to 307 and338 to 357 representing the regions showing homology to plant and fungalRNases (Schneider et al., 1993) are underlined.

[0158]FIG. 2: Rectal temperature curve of animals after test infection

[0159] Daily rectal temperature was recorded from day 2 before till day18 post infection. Rectal temperature curve is detailed for each animalof the group infected with the virus V(pA/CSFV) (continuous line)derived from plasmid pA/CSFV or with the virus V(pA/C-346-d) derivedfrom plasmid pA/C-346-d (dotted line).

[0160]FIG. 3: Rectal temperature curve of animals after challengeinfection

[0161] Daily rectal temperature was recorded at days 1-21 post challengevirus infection. Animals challenged with a lethal dosis of the CSFVchallenge strain Eystrup had been infected with mutant C-346-d[V(pA/C-346-d)] 69 days in before as detailed in the text. Rectaltemperature curve is detailed for each animal of the group challengedwith 2×10⁵ TCID₅₀ from the CSFV challenge strain Eystrup

[0162]FIG. 4: Rectal temperature curve of animals after test infection

[0163] Daily rectal temperature was recorded at days 0-18 postinfection. Rectal temperature curve is detailed for each animal of thetwo groups infected either with C-346-d [V(pA/C-346-d)] (dotted line) orwith the restored virus C-346-d/RS [V(pA/C-346-d/Rs)] (continuous line).

[0164]FIG. 5: Rectal temperature after challenge infection animalexperiment #2

[0165] Daily rectal temperature was recorded at days 1-10 post challengevirus infection. Animals challenged with a lethal dose (2×10⁵ TCID₅₀) ofthe CSFV challenge strain Eystrup had been infected with mutant C-346-d37 days in before.

[0166]FIG. 6: Rectal temperature of animals treated with a double mutantaccording to example 6

[0167] Daily rectal temperature was recorded prior and post challengevirus infection with mutant V(pA/C-297-L/346-L).

[0168]FIG. 7: Discrimination between C-346-d and CSFV without deletionof the histidine codon 346 by RT-PCR according to example 8

[0169] a) Primer pair Ol H−3/01 E^(ms)Stop allows to specificallyamplify a band derived from RNA containing the deletion of codon 346(C-346-d) as described in detail in example 8. In contrast, RNA, notcontaining said deletion does not interact with said primer pair (C-WT,C-346-L, C-346-K).

[0170] b) And c) The other two primer combinations (Ol H+2 and Ol H+3)amplify bands derived from RNA that do not contain the deletion of codon346 (Ol H+2 and Ol H+3). No band can be observed when RNA from the346-deletion mutant C-346-d is used as a template.

1. A live vaccine comprising a pestivirus, wherein the RNase activityresiding in glycoprotein E^(RNS) is inactivated.
 2. The vaccine of claim1, wherein said RNase activity is inactivated by deletions and/ormutations of at least one amino acid of said glycoprotein.
 3. Thevaccine according to claim 2, wherein said deletions and/or mutationsare located at the amino acids at position 295 to 307 and/or position338 to 357, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other strains, of saidglycoprotein.
 4. The vaccine according to any one of claims 1 to 3,wherein said RNase activity is inactivated by deletion or mutation ofthe amino acid at position 346, as described in FIG. 1 for the CSFVAlfort strain in an exemplary manner or corresponding thereto in otherstrains, of said glycoprotein.
 5. The vaccine according to any one ofclaims 1 to 4, wherein said RNase activity is inactivated by thedeletion of the histidine residue at position 346, as described in FIG.1 for the CSFV Alfort strain in an exemplary manner or correspondingthereto in other strains, of said glycoprotein.
 6. A vaccine accordingto any one of claims 1 to 5 comprising a BVDV pestivirus, wherein saidRNase activity is inactivated by the deletion of the histidine residueat position 346, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other BVDV strains, of saidglycoprotein.
 7. A pestivirus, wherein the RNase activity residing inglycoprotein E^(RNS) is inactivated by deletions and/or mutations of atleast one amino acid of said glycoprotein with the proviso that theamino acids at position 297 and/or 346, as described in FIG. 1 for theCSFV Alfort strain in an exemplary manner or corresponding thereto inother strains, of said glycoprotein are not lysine.
 8. The pestivirus ofclaim 7, wherein said RNase activity is inactivated by deletions and/ormutations located at the amino acids at position 295 to 307 and/orposition 338 to 357, as described in FIG. 1 for the CSFV Alfort strainin an exemplary manner or corresponding thereto in other strains, ofsaid glycoprotein.
 9. The pestivirus of claim 7 or 8, wherein said RNaseactivity is inactivated by deletion or mutation of the amino acid atposition 346, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other strains, of saidglycoprotein.
 10. The pestivirus according to any one of claims 7 to 9,wherein said RNase activity is inactivated by the deletion of thehistidine residue at position 346, as described in FIG. 1 for the CSFVAlfort strain in an exemplary manner or corresponding thereto in otherstrains, of said glycoprotein.
 11. A BVDV pestivirus according to anyone of claims 7 to 10, wherein said RNase activity is inactivated by thedeletion of the histidine residue at position 346, as described in FIG.1 for the CSFV Alfort strain in an exemplary manner or correspondingthereto in other BVDV strains, of said glycoprotein.
 12. A nucleic acidcoding for a glycoprotein E^(RNS), wherein the RNase activity residingin said glycoprotein is inactivated by deletions and/or mutations of atleast one amino acid of said glycoprotein with the proviso that theamino acids at position 297 and/or 346, as described in FIG. 1 for theCSFV Alfort strain in an exemplary manner or corresponding thereto inother strains, of said glycoprotein are not lysine.
 13. The nucleic acidof claim 12, wherein said RNase activity is inactivated by deletionsand/or mutations that are located at the amino acids at position 295 to307 and/or position 338 to 357, as described in FIG. 1 for the CSFVAlfort strain in an exemplary manner or corresponding thereto in otherstrains, of said glycoprotein.
 14. The nucleic acid of claim 12 or 13,wherein said RNase activity is inactivated by deletion or mutation ofthe amino acid at position 346, as described in FIG. 1 for the CSFVAlfort strain in an exemplary manner or corresponding thereto in otherstrains, of said glycoprotein.
 15. The nucleic acid according to any oneof claims 12 to 14, wherein said RNase activity is inactivated by thedeletion of the histidine residue at position 346, as described in FIG.1 for the CSFV Alfort strain in an exemplary manner or correspondingthereto in other strains, of said glycoprotein.
 16. A BVDV nucleic acidaccording to any one of claims 12 to 15, wherein said RNase activity isinactivated by the deletion of the histidine residue at position 346, asdescribed in FIG. 1 for the CSFV Alfort strain in an exemplary manner orcorresponding thereto in other BVDV strains, of said glycoprotein. 17.Use of nucleic acids according to any one of claims 12 to 16 forpreparing nucleotide- and/or vector-vaccines.
 18. A pharmaceuticalcomposition comprising a vaccine according to any one of claims 1 to 6,and/or a pestivirus according to any one of claims 7 to 11, and/or anucleotide sequence according to any one of claims 12 to
 16. 19. Amethod for attenuating pestiviruses characterized in that the RNaseactivity residing in glycoprotein E^(RNS) is inactivated.
 20. The methodof claim 19, wherein said RNase activity is inactivated by deletionsand/or mutations of at least one amino acid of said glycoprotein. 21.The method of claim 19 or 20, wherein said deletions and/or mutationsare located at the amino acids at position 295 to 307 and/or position338 to 357, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other strains, of saidglycoprotein.
 22. The method according to any one of claims 19 to 21,wherein said RNase activity is inactivated by deletion or mutation ofthe amino acid at position 346, as described in FIG. 1 for the CSFVAlfort strain in an exemplary manner or corresponding thereto in otherstrains, of said glycoprotein.
 23. The method according to any one ofclaims 19 to 22, wherein said RNase activity is inactivated by thedeletion of the histidine residue at position 346, as described in FIG.1 for the CSFV Alfort strain in an exemplary manner or correspondingthereto in other strains, of said glycoprotein.
 24. A method forproducing a specifically attenuated vaccine characterized in that theRNase activity residing in glycoprotein E^(RNS) is inactivated.
 25. Themethod of claim 24, wherein said RNase activity is inactivated bydeletions and/or mutations of at least one amino acid of saidglycoprotein.
 26. The method of claim 24 or 25, wherein said deletionsand/or mutations are located at the amino acids at position 295 to 307and/or position 338 to 357, as described in FIG. 1 for the CSFV Alfortstrain in an exemplary manner or is corresponding thereto in otherstrains, of said glycoprotein.
 27. The method according to any one ofclaims 24 to 26, wherein said RNase activity is inactivated by deletionor mutation of the amino acid at position 346, as described in FIG. 1for the CSFV Alfort strain in an exemplary manner or correspondingthereto in other strains, of said glycoprotein.
 28. The method accordingto any one of claims 24 to 27, wherein said RNase activity isinactivated by the deletion of the histidine residue at position 346, asdescribed in FIG. 1 for the CSFV Alfort strain in an exemplary manner orcorresponding thereto in other strains, of said glycoprotein.
 29. Amethod for detectably labeling pestiviruses characterized in that theRNase activity residing in glycoprotein E^(RNS) is inactivated.
 30. Themethod of claim 29, wherein said RNase activity is inactivated bydeletions and/or mutations of at least one amino acid of saidglycoprotein.
 31. The method of claim 29 or 30, wherein said deletionsand/or mutations are located at the amino acids at position 295 to 307and/or position 338 to 357, as described in FIG. 1 for the CSFV Alfortstrain in an exemplary manner or corresponding thereto in other strains,of said glycoprotein.
 32. The method according to any one of claims 29to 31, wherein said RNase activity is inactivated by deletion ormutation of the amino acid at position 346, as described in FIG. 1 forthe CSFV Alfort strain in an exemplary manner or corresponding theretoin other strains, of said glycoprotein.
 33. The method according to anyone of claims 29 to 32, wherein said RNase activity is inactivated bythe deletion of the histidine residue at position 346, as described inFIG. 1 for the CSFV Alfort strain in an exemplary manner orcorresponding thereto in other strains, of said glycoprotein.
 34. Amethod for the prophylaxis and treatment of pestivirus infections inanimals characterized in that a vaccine according to any one of claims 1to 6 or a pharmaceutical composition according to claim 18 is applied toan animal in need of such prophylaxis or treatment.
 35. A process forthe preparation of specifically attenuated pestiviruses characterized inthat the RNase activity residing in glycoprotein E^(RNS) is inactivated.36. The process according to claim 35, wherein said RNase activity isinactivated by deletions and/or mutations of at least one amino acid ofsaid glycoprotein.
 37. The process according to claim 35 or 36, whereinsaid deletions and/or mutations are located at the amino acids atposition 295 to 307 and/or position 338 to 357, as described in FIG. 1for the CSFV Alfort strain in an exemplary manner or correspondingthereto in other strains, of said glycoprotein.
 38. The processaccording to any one of claims 35 to 37, wherein said RNase activity isinactivated by deletion or mutation of the amino acid at position 346,as described in FIG. 1 for the CSFV Alfort strain in an exemplary manneror corresponding thereto in other strains, of said glycoprotein.
 39. Theprocess according to any one of claims 36 to 38, wherein said RNaseactivity is inactivated by the deletion of the histidine residue atposition 346, as described in FIG. 1 for the CSFV Alfort strain in anexemplary manner or corresponding thereto in other strains, of saidglycoprotein.
 40. A process for the preparation of specifically labeledpestiviruses characterized in that the RNase activity residing inglycoprotein E^(RNS) is inactivated.
 41. The process according to claim40, wherein said RNase activity is inactivated by deletions and/ormutations of at least one amino acid of said glycoprotein.
 42. Theprocess according to claim 40 or 41, wherein said deletions and/ormutations are located at the amino acids at position 295 to 307 and/orposition 338 to 357, as described in FIG. 1 for the CSFV Alfort strainin an exemplary manner or corresponding thereto in other strains, ofsaid glycoprotein.
 43. The process according to any one of claims 40 to42, wherein said RNase activity is inactivated by deletion or mutationof the amino acid at position 346, as described in FIG. 1 for the CSFVAlfort strain in an exemplary manner or corresponding thereto in otherstrains, of said glycoprotein.
 44. The process according to any one ofclaims 40 to 43, wherein said RNase activity is inactivated by thedeletion of the histidine residue at position 346, as described in FIG.1 for the CSFV Alfort strain in an exemplary manner or correspondingthereto in other strains, of said glycoprotein.
 45. Use of a vaccine ofany one of claims 1 to 6 for the prophylaxis and treatment of pestivirusinfections in animals.
 46. Use of a pharmaceutical composition of claim18 for the prophylaxis and treatment of pestivirus infections inanimals.
 47. Use of a pestivirus of any one of claims 7 to 11 and/or anucleotide sequence according to any one of claims 12 to 16 for thepreparation of a vaccine or a pharmaceutical composition.
 48. A methodfor distinguishing pestivirus-infected animals from animals vaccinatedwith a specifically attenuated pestivirus, wherein said specificallyattenuated pestivirus is attenuated according to a method of any one ofclaims 19 to 23, comprising the following steps: (1) Obtaining a samplefrom an animal of interest suspected of pestivirus infection or avaccinated animal; (2) Identifying the nucleotide sequence of apestivirus within said sample; (3) Correlating the deletions and/ormutations of the E^(RNS) nucleotide sequence as present in the vaccinewith a vaccinated animal and correlating the absence of said deletionsand/or mutations with a pestivirus infection of said animal.
 49. Themethod of claim 48, comprising the following steps: (1) Obtaining asample from an animal of interest suspected of pestivirus infection or avaccinated animal; (2) Identifying a modified E^(RNS) glycoprotein of anattenuated pestivirus by the specific binding of monoclonal orpolyclonal antibodies to E^(RNS) glycoproteins present in said sample,said glycoproteins being modified by a method according to any one ofclaims 19 to 23, whereby said monoclonal or polyclonal antibodies do notbind to unmodified E^(RNS) glycoproteins; (4) Correlating the specificbinding of said monoclonal or polyclonal antibodies with a vaccinatedanimal and correlating the absence of antibody binding to a pestivirusinfection of said animal under the proviso that the presence ofpestiviral material in said animal and/or said sample is establishedotherwise.
 50. The method of claim 49, comprising the following steps:(1) Obtaining a sample from an animal of interest suspected ofpestivirus infection or a vaccinated animal; (2) Identifying anunmodified E^(RNS) glycoprotein of a pestivirus by the specific bindingof monoclonal or polyclonal antibodies to E^(RNS) glycoproteins presentin said sample, said glycoproteins not being modified by a methodaccording to any one of claims 19 to 23, whereby said monoclonal orpolyclonal antibodies do not bind to modified E^(RNS) glycoproteins; (3)Correlating the specific binding of said monoclonal or polyclonalantibodies with a pestivirus infection in said animal and correlatingthe absence of antibody binding to an vaccinated animal under theproviso that the presence of pestiviral material in said animal and/orsaid sample is established otherwise.
 51. The method of claim 48,comprising the following steps: (1) Obtaining a sample from an animal ofinterest suspected of pestivirus infection or a vaccinated animal; (2)Determining the absence or presence of RNase activity of a glycoproteinE^(RNS) within said sample; (3) Correlating the absence of RNaseactivity of glycoprotein E^(RNS) with a vaccinated animal andcorrelating the presence of said activity with a pestivirus infection ofsaid animal.
 52. The method of claim 48, comprising the following steps:(1) Obtaining a sample of polyclonal antibodies from an animal ofinterest suspected of pestivirus infection or a vaccinated animal; (2)Identifying any specific binding of said polyclonal antibodies tounmodified glycoprotein E^(RNS) or glycoprotein E^(RNS) as modifiedaccording to the invention. (3) Correlating the binding of saidpolyclonal antibodies to unmodified glycoprotein E^(RNS) with apestivirus infection and correlating the binding of said polyclonalantibodies to glycoprotein E^(RNS) as modified according to theinvention with a vaccinated.